The excessive desmoplasia is the hallmark of human pancreatic cancer that influences the local T-cell-based immune response. In the present work, the stromal collagen organization in normal and malignant pancreatic tissues as well as its relationsship to T-cell distribution in pancreatic cancer were studied. It was found that differences in collagen organization do not change the spatial orientation of T-cell migration and do not influence the availability of tumor cells for T-cells. The results of the study do not support the concept of use of stroma collagen organization for improvement of spatial T-cell distribution in the tumor. The dominant intrastromal T-cell infiltration in pancreatic cancer is mainly caused by the contact guidance through the excessive desmoplastic reaction and could represent one of the obstacles to an effective immune response in this tumor type. This study analyzed the collagen organization in normal and malignant pancreatic tissues as well as its influence on T-cell distribution in pancreatic cancer. Human pancreatic tissue was analyzed using immunofluorescence staining and multiphoton and SHG microscopy supported by multistep image processing. The influence of collagen alignment on activated T-cells was studied using 3D matrices and time-lapse microscopy. It was found that the stroma of malignant and normal pancreatic tissues was characterized by complex individual organization. T-cells were heterogeneously distributed in pancreatic cancer and there was no relationship between T-cell distribution and collagen organization. There was a difference in the angular orientation of collagen alignment in the peritumoral and tumor-cell-distant stroma regions in the pancreatic ductal adenocarcinoma tissue, but there was no correlation in the T-cell densities between these regions. The grade of collagen alignment did not influence the directionality of T-cell migration in the 3D collagen matrix. It can be concluded that differences in collagen organization do not change the spatial orientation of T-cell migration or influence stromal T-cell distribution in human pancreatic cancer. The results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of T-cell–tumor cell contact in pancreatic ductal adenocarcinoma.  Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a very poor prognosis . StandarStrong desmoplasia is a hallmark of pancreatic cancer , which cT-cell movement through the three-dimensional (3D) extracellular matrix (ECM) occurs via a process of amoeboid-like migration . It is dOur previous study investigated the mechanisms controlling intratumoral T-cell migration in PDAC in terms of chemokines and contact guidance and determined whether and how these mechanisms contribute to immune evasion of pancreatic cancer . AdditioGenerally, fibrillar collagen is highly abundant in the stroma of pancreatic cancer ,19,20. TIt has previously been shown that the alignment of collagen fibers is an important factor that controls the contact guidance of tumor cells ,26. In tIn the present study, we defined individual stroma regions in PDAC patients based on the fibrillar collagen network. Subsequently, the newly established procedure of image processing and analysis enabled, for the first time, the generation of tailored data on collagen organization and T-cell distribution in two individually defined PDAC regions. Tissue samples of the normal pancreas, chronic pancreatitis, PDAC, and ACC were obtained from the tissue collection (PancoBank) of the European Pancreatic Center (EPZ), University of Heidelberg. Patients admitted to the study were undergoing surgery for pancreatic diseases in the Department of Surgery at the University of Heidelberg. The protocol was approved by the local ethics committee and informed consent was obtained according to the Helsinki declaration. Tissue samples were snap-frozen and stored in liquid nitrogen. Hematoxylin and eosin staining of 7 µm slides was performed using Meyer’s hemalaun  and Eosin G . The histological diagnosis and grading of single tumor samples were performed by a specialist in pancreatic pathology (F.B.). The tumor cellularity of PDAC samples was analyzed as previously described . It was All histological and immunofluorescence stainings were performed using filtered particle-free solutions and under sterile conditions, which ensured that optical artefacts did not appear in the tissue slides. For immunohistochemistry, 7 or 100 µm (for 3D reconstruction) acetone-fixed cryosections were pre-blocked using 20% goat serum  for 30 min and stained using rabbit anti-human Cytokeratin 7  and mouse anti-human CD3  followed by Alexa Fluor 568-conjugated secondary anti-mouse IgG . All incubations of labeled antibodies were performed in the dark for 1 h at room temperature. The staining procedures of 7 and 100 µm tissue slides were identical. Respective isotypic antibodies (Biolegend) were used as controls for immunofluorescence staining. A TriM Scope 2-photon microscope  mounted on a Nikon FN-1 upright stand  and equipped with a water-dipping 16× NA 0.8 long working distance objective  was used. The excitation was carried out with a Chameleon Ultra II femtosecond titan:sapphire laser   at 800 nm for collagen and 940 nm for Alexa Fluor 488-labeled epithelial cells and 850 nm for Alexa Fluor 568-labeled T-cells. The forward SHG signal of the collagen fibers was detected with a 405/10 nm bandpass filter. The emission of the labeled tumor clusters was detected at 525/50 nm and the emission of the labeled T-cells at 605/50 nm. Individual images of 625 µm × 625 µm  were acquired with a 20% overlap between images. The interval between Z-stack images was set on 1.0 μm. Three-dimensional reconstructions of stitched stacks were created using arivis Vision4D software .Image files of SHG, tumor, and T-cell images were renamed (macro RenameInputFile) in ImageJ plugin  and images were stitched (macro StitchingData) to create fused maximum intensity projection (MIP) images as well as fused Z-stacks. Prior to quantification of the collagen fibers, fused SHG MIP images were transformed into 8-bit images and adjusted using threshold between 10 and 255 gray levels to eliminate background noise. CT-FIRE and CurveAlign  were run on fused MIP images according to the manufacturer’s instructions to calculate the collagen fiber parameters: length, number, straightness, and width (CT-FIRE V1.3); fiber alignment (CurveAlign V3.0), as previously described ,35. Fibe2. Indeed, the binary tumor MIP were used for the individual collagen analysis in the tumor microenvironment and the binary MIP of the T-cells for the individual T-cell distribution analysis inside and outside the peritumoral stroma regions of PDAC samples. The analysis steps are summarized in the ImageJ macros code  and in the Tumor cluster and T-cell segmentation was performed using a combination of the pixel classification workflow based on the machine-learning image analysis (ilastik)  and ImagImages of tumor masks were generated by manually reconstructing the tumor clusters in fused SHG–MIP images using CurveAlign V4.0. The mean collagen fiber length in the PDAC samples was calculated to be 22.4 µm using CT-FIRE V1.3. Using these tumor mask images in combination with the previously generated CT-FIRE results of the average collagen fiber length in PDAC, a detailed, individual description of collagen in the PDAC microenvironment of twelve (see 4.4) selected PDAC tissues samples was generated. The mean fiber length (22.4 µm) was used as the interval by which the distance to microscopic tumor borderline was segmented. The analysis was performed in an individual area up to a maximal distance of 246.4 µm (eleven segments/areas of 22.4 µm). In these eleven areas, the angular distribution of the collagen fibers was analyzed and the relative fraction (R) of fibers having an angular range from 0° to 15°  was calculated. The ratio between individual R values and the mean (plateaued) R value from five distant areas  using the scale: 0 = absent, 1 = low, 2 = moderate, 3 = high, and 4 = very high infiltration, as previously described  Figure . PDAC G1After identification of the expansion of individual peritumoral stroma regions, peritumoral and tumor-cell-distant ROIs of binary images of T-cell reconstructions were generated in ImageJ (macro DrawRectangularROIs). The number of generated ROIs varied between 2 and 12 for both peritumoral and tumor-cell-distant ROIs per SHG image. The size of the generated ROIs corresponded to the X and Y dimensions of the individual peritumoral stroma region. The collagen fibers of the individual ROIs were analyzed using CT-FIRE and CurveAlign and the mean values of the five collagen parameters were determined for each PDAC tissue. The T-cell area in each ROI was measured in ImageJ in an automated way. PDAC specimens showing distinct T-cell infiltration were used for the analysis. The total time required for analysis of single images was very different. Twelve images (12 different patients) requiring the lowest time for image processing were selected for the analysis. The areas of the individual peritumoral stroma regions of PDAC images and the areas of the tumor clusters in the peritumoral and tumor-cell-distant stroma regions were determined in ImageJ. In the binary images of T-cell reconstructions, the total area of CD3+ T-cells per image and the area of CD3+ T-cells inside and outside the individual peritumoral stroma regions were determined. The T-cell coefficients and T-cell densities of the two PDAC stroma regions were calculated using the four formulas below: 2. Isolated peripheral blood mononuclear leukocytes (PBMLs) were isolated from the human blood of healthy donors using density gradient centrifugation with Biocoll . The cells were routinely cultivated in RPMI 1640  supplemented with 10% fetal calf serum (FCS) (cc-pro), 2 mM L-glutamine, 20 U/mL penicillin, 0.1 mg/mL streptomycin (cc-pro), 1 mM sodium pyruvate , and 0.05 mM β–mercaptoethanol  at 37 °C in a humidified atmosphere with 5% COFor activation, PBMLs were stimulated with 12.5 µg/mL Concanavalin A  and 50 U/mL interleukin-2 (IL-2)  for day 1, and further with IL-2 for days 2–6. The activated cells were collected and used on day 6. To obtain the CD3+ population, mainly consisting of T-cells, the cells were labeled with PE-conjugated anti-CD3 mAb  and isotypic mAb  was used as a control. The percentage of CD3+ cells was determined using a flow cytometer  and FlowJo V.10.1 software . The fraction of CD3+ cells was 70–80% . 5/well) of HTS Transwell-96 plates . Migration was stimulated using 0–10,000 ng/mL SDF-1α, MIP-1β, or IP-10  and measured after 3 h as fluorescence intensity in the lower wells using a spectrofluorimeter . This experiment was repeated four times. For Boyden chamber assays, phenol red-free RPMI medium  supplemented with 1 mM sodium pyruvate , 0.03 mM bovine serum albumin , 2 mM L-glutamine, 20 U/mL penicillin, 0.1 mg/mL streptomycin (cc-pro), and 0.05 mM 2-mercaptoethanol (Life Technologies) was used. The cells were labeled for 45 min at 37 °C with 4 µM calcein AM  and seeded in the upper wells , supplemented with bovine serum albumin fraction V  and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 0.34 M sodium hydroxide, FITC-conjugated bovine collagen type I (Sigma-Aldrich), rat collagen type I , and magnetic beads   followed by Alexa Fluor 568-conjugated secondary anti-mouse IgG (Abcam). Approximately 1 × 10MA, USA) . The fin2) was performed for 30 min. The experiments were repeated six times, from which the data of T-cell migration without SDF-1α  and with SDF-1α  were collected. For the recordings of the collagen matrices as well as the time-lapse videos of the T-cells, the Nikon A1R confocal microscope  with a 20× immersion-free objective  was used. For imaging, the microfluidic chamber was placed into the temperature-controlled (37 °C) chamber . Fluorescent collagens and T-cells were detected using excitations of 488 and 525 nm, and emissions of 525 and 595 nm, respectively. Time-lapse recording (2 images/min) of four neighboring fields . The axial orientation of T-cell migration was measured as an integral value from single steps of individual T-cell trajectories. CT-FIRE and CurveAlign were run on fused confocal images to describe the collagen parameter in the in vitro model. Dependent on the local alignment coefficient, the collagen matrix was assigned to one of three groups: non-aligned, partially aligned, or aligned collagen .t-test and Bonferroni–Holm correction. Differences in individual collagen parameters  between different groups were compared using the Mann–Whitney U test and Bonferroni–Holm correction. A value of p < 0.05 was considered significant. Pearson’s correlation coefficient (r) was used to measure the strength of linear association between T-cell density and parameters of collagen organization in tissue as well as between collagen alignment and axial orientation of T-cell migration in vitro. Statistical analysis was performed using the SPSS software . The alignment coefficient of collagen fibers in the pancreatic tissue was compared using the The SHG microscopy images showed perilobular collagen in the connective tissue A,B and hT-cells rarely infiltrated pancreatic tissues and formed local (mainly perivascular) accumulations of grades 1–2, and in a few cases of grades 3–4 . T-cells were mainly accumulated in perivascular areas and regions of loosened collagen C,D. Generally, the stroma of PDAC was characterized by a very heterogeneous collagen network with morphologically distinct collagen forms and organization patterns A–D. The Intratumoral infiltration of grades 2–4 were found in pancreatic cancers C,E. PDACFor the analysis of the spatial relationship between collagen, tumor cells, and T-cells, 3D reconstructions of multiphoton and SHG images were used. It was clearly demonstrated that T-cell infiltration was mainly localized in the stroma although the direct contact between T-cell and tumor cells could also be (in 23 of 32 samples) identified E. Two of three ACC samples showed minimal stroma having short collagen fibers with heterogeneous morphology E. In oneACCs were ubiquitously infiltrated with T-cells. Two samples showed moderate (grade 2), and one sample showed severe (grade 3) infiltration. Although direct T-cell–tumor cell contact, as well as tumor cluster-infiltrating T-cells, can frequently be found, the vast majority of infiltrating T-cells were localized in the stroma E. p < 0.05  CD3 expression . The T-cIn the present study, the collagen organization in normal pancreas, chronic pancreatitis, and pancreatic cancer and its potential influence on the distribution of infiltrating T-cells was investigated. Two features of collagen organization, mean fiber diameter and straightness, were at the same range between different tissue types and also showed low individual deviations. The mean fiber length was also at the same range, although single fibers were very long. Because there were no differences, the above-mentioned parameters can hardly be expected to change T-cell migration. Other features, such as collagen density, alignment, and spatial orientation, showed higher individual heterogeneity and are discussed below. In a normal pancreas tissue, the stroma is formed during body growth and no significant remodeling is expected under healthy conditions. Therefore, stromal collagen in a normal pancreas displays a well-aligned and generally homogeneous structure of any determined parameters. Chronic pancreatitis showed the same homogeneous collagen organization as a normal pancreas. The microenvironmental changes in chronic pancreatitis develop slowly and, therefore, the collagen network in progressing fibrosis follows the same organization patterns as in the normal pancreas. In contrast to the normal pancreas, pancreatic cancer (PDAC and ACC) showed very heterogeneous patterns of collagen organization, although some common principles have also been found. Both PDAC and ACC may be considered as dynamic systems with continuously changing microenvironments, including spatial extension and active ECM production and remodeling. As expected, it had consequences for the collagen organization and resulted in an inhomogeneous collagen presentation, especially for fiber length, collagen density, and alignment. All these parameters could be important as a mechanical barrier for leukocytes; however, no significant differences in mean values between the normal pancreas, chronic pancreatitis, and pancreatic cancer were found. The only significantly increased collagen alignment observed was in chronic pancreatitis and may be the result of the high collagen productivity of cells in the fibrotic tissues. A group of PDAC samples showed a ubiquitous collagen organization, whereas other samples showed highly individual patterns. It could only reflect the inhomogeneity of local microenvironmental conditions (see above) influencing collagen production. Previous studies have analyzed the prognostic relevance of collagen organization and related it to changes in tumor invasion; in PDAC, high collagen alignment has been shown to be a negative prognostic factor . IncreasPrevious studies have established that pancreatic cancer induces T-cell immune responses; however, infiltrating T-cells are mainly “trapped” in the tumor stroma . This deAmong the different parameters of collagen organization, collagen alignment and its directional orientation were proposed to be important factors that could guide T-cell migration and distribution. In the present study, only the specific angular ranges of collagen fibers were dependent on the distance to the tumor stroma border by which peritumoral and tumor-cell-distant stroma regions were determined. We propose that this difference can mainly be caused by stroma compression through the spatial expansion of tumor cells. Interestingly, no relationship was found between the T-cell densities in the peritumoral and tumor-cell-distant stroma regions. It demonstrates that the spatial orientation of collagen did not change T-cell distribution in the stroma of pancreatic cancer. T-cell migration was also analyzed in differently aligned and differently dense collagens in an in vitro model. Although the phenotype of in vitro activated T-cells is different to that of tumor-infiltrating lymphocytes, this model could be representative of studies of the mechanical interaction between aligned collagen fibers and migrating T-cells. The rigid nucleus defines the smallest size of gaps or pores that can be breached by T-cells during migration ,41. The Recently, Pruitt et al. reported a study on the guidance of activated T-cells by aligned collagen . In thisFinally, the present study described the unique workflow, which included a complex of histological, microscopic, and image processing operations. The above operation algorithm enabled a more accurate analysis of pancreatic stroma collagen organization compared to the ones reported in any previous studies ,46. The The low number of tissue samples could be a limitation of the present study; however, the total PDAC cohort (n = 32) should be sufficient for the characterization of collagen organization in this tumor type. Despite the use of machine-learning-based bioimage analysis, the workflow part of pixel classification and area analysis was a time- and resource-consuming procedure that in the present study could only be performed on a limited sample number. Nevertheless, the final number (n = 12) provided the information with the highest accuracy, and the data were conclusive for the described analyses. To summarize, in contrast with migrating tumor cells, collagen alignment does not force T-cells into directional migration, nor does it influence intratumoral T-cell distribution. From this point of view, the results of the present study do not support the rationale of remodeling of stroma collagen organization for improvement of direct T-cell–tumor cell contact in PDAC. The proposed tissue and image processing and analysis workflow may represent an excellent tool for the measurement of individual collagen organization in cancer patients.


There is a growing concern that the use of anti-hypertensives may be associated with an increased risk of cancer, but it remains uncertain for the association between anti-hypertensives and lung cancer risk, as well as their interaction with aspirin in chemoprotective effects.β-blocker,α-blocker) and/or aspirin were included as the exposure groups. Using the Cox proportional hazards model with inverse probability weighting, we estimated hazard ratios (HRs) with 95% confidence intervals (CIs) for lung cancer risk from anti-hypertensives usage or combination usage of aspirin with anti-hypertensives. The likelihood ratio test and interaction model were adopted for exploring the interaction effects with aspirin.The goal of this study is to assess the association between anti-hypertensives use and the risk of lung cancer, as well as the chemopreventive impacts from the combination usage of aspirin and anti-hypertensives. A retrospective cohort study was conducted based on all the public hospital electronic medical records in Hong Kong. Patients with prescription records of anti-hypertensives . Moreover, the lower risk of lung cancer persisted with a longer follow-up period of anti-hypertensives usage. Combination usage with aspirin in the users of ACEi/ARB, CCB, and α-blocker showed significant interaction effects. However, the smoking effect could not be eliminated in this analysis.A total of 6592 and 84,116 lung cancer cases were identified from the groups of anti-hypertensives users and anti-hypertensives users with aspirin, respectively. The group of non-aspirin patients who received anti-hypertensives showed a significantly lower risk of lung cancer , compared to those without anti-hypertensivesAnti-hypertensive treatment was associated with a lower risk of lung cancer, which is associated with the anti-hypertensives exposure period. The potential interaction on the chemopreventive influence from combination usage of α-blocker and aspirin might exist. More corroborations on these findings are needed to focus on the different settings in future studies.The online version contains supplementary material available at 10.1186/s12885-021-08971-6. Lung cancer has been the leading cause of cancer death worldwide, accounting for an estimated 1.76 million cases in 2018 . MultiplAlpha-adrenergic receptor blockers (α-blockers), and Beta-adrenergic receptor blockers (β-blockers) [β-blockers and ACEi (46 and 19% respectively) among all the prescription anti-hypertensive agents [The antihypertensives include angiotensin I-converting enzyme inhibitors (ACEis), angiotensin II receptor blockers (ARBs), calcium channel blockers (CCBs), diuretics, lockers) . In Honglockers) .. A stude agents . ConsideAs an aging society with an elderly population expected to double over the next 25 years, the burden of cancer and cardiovascular (CVD) in Hong Kong is predicted increase dramatically. In light of the conflicting and limited evidence from Asian population on the long-term anti-hypertensives use and lung cancer risk, a large population-based cohort study was conducted . We aimeThis is a retrospective cohort study which enrolled long-term aspirin and antihypertensive drugs users with 10 years of follow-up within electronic medical records in all the public hospitals of Hong Kong. The data records could represent the whole population of aspirin usage due to the reason that the Hong Kong Hospital Authority from Hong Kong government supports the long-term care for citizens who have chronic diseases with little expense for patients. Thus, the database in this study is representative of the population that had chronic diseases while maintaining aspirin and anti-hypertensives usage.In addition, our study followed the standard guidelines of Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). All medical records were obtained anonymously and patients were assigned anonymous identification code as required by the policy of Hong Kong Hospital Authority from Hong Kong government. As a result, the consent to participate was not applicable and the informed consent was waived, by the ethical approval reached from the Joint Chinese University of Hong Kong - New Territories East Cluster Clinical Research Ethics Committee (CREC ref. no.:2014.461).N = 602,936). Participants who were diagnosed with cancer according to the ICD codes were extracted from Hong Kong electronic health system. Individuals with prevalent cancer at the start of follow up were excluded, but individuals with cancer found in different locations in the follow-up duration were included in the study. Moreover, all the patients’ data extraction for academic institutions are under the strict supervision and regulation for protection of privacy.From the Hospital Authority Clinical Data Repository, among the 4,564,100 eligible study participants who had ever used any public healthcare service between 2000 and 2004, subjects aged over 18 years old with aspirin/anti-hypertensives prescription and the age-and-gender-matched control group with non-aspirin at the ratio of 1:2 were included in our original study [α-blocker also belongs to the main therapy in men with lower urinary tract symptom, the only α-blocker drugs intake group just included the male patients in our study. Moreover, ACEi and ARB shared the similar mechanism in the renin-angiotensin-aldosterone system (RAAS) pathway that is widely implicated in diabetes (DM), hypertension, and heart failure, so they were analysed as a group of ACEi/ARB. The usage of diuretics only was excluded as it was also widely used for other symptoms such as oedema due to different underlying causes.Participant follow-up time was measured from the time of cohort entry, until the date of death or end of 2013, whichever came first. The medication and disease diagnosis records were kept tracking for each participant. Index date was defined as the first prescription record of aspirin or anti-hypertensives drugs. The durations of aspirin and anti-hypertensives prescription histories were computed for each participant by calculating the first and last prescriptions time. Among them, patients who had taken aspirin and anti-hypertensive drugs for less than 6 months were excluded. If a patient suspended the medication prescription temporarily, the gap period of no aspirin/anti-hypertensives usage was also calculated. Generally, the aspirin prescription in Hong Kong is mainly to prevent cardiovascular and cerebrovascular diseases, so patients with comorbidities of CVDs, including ischemic heart diseases (IHD) (ICD-9: 410–414), stroke , and other cerebrovascular diseases (ICD-9: 430–438), were identified from the electronic health system. Additionally, we also documented the other medications usage including antiplatelet agents (apart from aspirin), non-steroidal anti-inflammatory drugs (NSAID), anticoagulant  and other antisecretory agents such as proton pump inhibitor (PPI) or H2-antagonist (H2B) [P-value < 0.05 was adopted to define statistical significance. All the analyses were performed in the R package version 3.6.1.The frequencies with percentages for categorical variables, medians with interquartile ranges (IQR), or means with standard deviations (SD) for continuous variables were calculated during the whole cohort follow-up period. We adopted the Cox proportional hazards regression models with inverse probability (IP) to assess the hazard ratios (HR) with 95% confidence intervals (CIs) for the association between different medication usages of 1) aspirin usage, 2) aspirin and anti-hypertensives combination usage, and lung cancer risks. The different timescales for the follow-up time points included the years of 2006, 2009, and 2013. IP weighting was used to adjust for confounding factors including NSAID, H2 blocker, anticoagulant, antiplatelet, PPI, statin, as drug usages can indirectly reflect comorbidities of patients. Then the likelihood ratio test was adopted to compare the additive model and the interaction model, and the interaction effects with the cross-classification by anti-hypertensives and aspirin usage were explored. In addition, follow-up and subgroup analyses were performed by different follow-up period, gender, DM, and age groups. Sensitivity analysis with different drugs exposure period were also conducted to evaluate the robustness of findings. All models were adjusted for the following variables measured at cohort entry: age, sex, comorbidities, and other medications. Among all the 4,564,100 patients who came to visit the clinics or hospitals under the Hospital Authority internal network during the period of 2000–2004, there were 745,967 subjects who were identified as aspirin users and its matched control group. After applying the exclusion criteria, the final total sample size for this study was 602,936  and aspirin & anti-hypertensives separately (3.3%) respectively. Additional characteristics for the group of anti-hypertensives users and non-anti-hypertensives users are also shown in For the participants enrolled in the study, the mean age was 67.5 SD = 12.0) years old. Table 2.0 yearsn = 6592; 2.8%), only aspirin users , and combination of anti-hypertensives and aspirin users . Table β-blocker , and α-blocker  were associated with a statistically significant lower risk of lung cancer.Overall, the lung cancer incidences for different group were displayed in Table α-blocker with aspirin  with significant interaction (P = 0.008). However, no significant interaction effects were identified between all anti-hypertensives and aspirin usage (P = 0.090).Interactive model was adopted. After the adjustment of age, sex, comorbidities, and medications, the significant interaction effects persisted among the combination treatment of ACEi/ARB with aspirin, CCB with aspirin, as well as α-blocker were associated with a solidly lower risk of lung cancer. With the follow-up period up to 7, 10, and 14 years, the HRs of lung cancer for single ACEi/ARB users were 0.55 (95%CI: 0.36–0.83), 0.53 (95% CI: 0.36–0.79), and 0.45 (95%CI: 0.32–0.62); and the HRs for α-blocker users were 0.70 (95%CI: 0.57–0.86), 0.68 (95% CI: 0.55–0.84), and 0.61 (95%CI: 0.48–0.77) respectively. When the aspirin was given concurrently, lower lung cancer risk remained for the users with α-blocker and aspirin medications for all the three follow-up periods. Concurrent use of α-blocker and aspirin lowered lung cancer risk by 57% (95%CI: 0.24–0.78), 59% (95%CI: 0.25–0.69), and 47% (95%CI: 0.34–0.84) for the follow-up period up to 7, 10, and 14 years respectively. Moreover, the trend test indicated that further reduced risk of lung cancer with longer use of anti-hypertensive drugs  to evaluate the impacts of the duration of medication usage to the lung cancer risk (Table Age significantly affected lung cancer risk (α-blocker demonstrated effects on lowering lung cancer risk (P < 0.001) among the patients who were older than 65 years old. Use of ACEi/ARB showed to have lower lung cancer risk by 56% , while use of α-blocker reduced lung cancer risk by 39%  among male patients. There was a significantly lower risk of lung cancer with anti-hypertensives usage regardless of DM status (P < 0.001). Among the patients without DM, significantly lower lung cancer risk was observed with the use of ACEi/ARB or α-blocker (P < 0.001) but not with concurrent aspirin use.cer risk . For oldM status . Among tP < 0.001). For these different treatment durations, the estimated HR of lung cancer were 0.64 (95%CI: 0.61–0.68), 0.67 (95%CI: 0.64–0.71), and 0.70 (95%CI: 0.66–0.74), which were non-significant statistically difference from the overall estimates in the main analysis. When combination usage of anti-hypertensives and aspirin was defined with at least one year use, it could reduce 33% lung cancer risk  which is also comparable to the main result with six-month exposure.We extended the minimum drugs treatment period to 1 year, 1.5 years, and 2 years from the original 6 months . The estFrom this large retrospective cohort study among Chinese population, neither the single anti-hypertensives treatment nor aspirin combination usage was observed to have excess of lung cancer risk. To the best of our knowledge, this is the first study to explore the lung cancer risk from combination usage of aspirin and anti-hypertensives.P > 0.05). Another study including 992,061 patients who were newly treated with anti-hypertensives showed that the ACEi treatment was associated with an increased risk of lung cancer by 14% (95%CI: 7–33%) [A network meta-analysis which included 70 randomised controlled trials reported no significant increased risk of cancer with the ACEi/ARB usage . Moreove: 7–33%) . A studyα-blocker and aspirin showed better chemopreventive impacts on the lung cancer risk for the patients who had no DM. The results were also consistent with the main findings from the overall cohort, while the consistent trend was not identified from other specific anti-hypertensives when they were used with aspirin synchronously. This might be due to some common molecular mechanisms in the chemotherapy pathway for the treatment of aspirin and antihypertensive, such as the enzyme cyclooxygenase (Cox) inhibition [Aside from the numerous epidemiological studies addressing the association between anti-hypertensives/aspirin to the risk of cancer imitation, promotion and recurrence, more studies on molecular pathway and mechanisms were conducted to address the conflicting results from epidemiological studies , 18. Unthibition . Moreovehibition , which mThe strengths of this study include 1) large study population in Hong Kong, 2) long follow-up period, and 3) different classes of anti-hypertensives studied. Despite such, some limitations should still need to be noticed. First, smoking status is the major confounder for the risk of lung cancer, but our hospital records did not include the history of smoking among the users of aspirin and anti-hypertensives. Previous studies demonstrated that patients with CVD may encourage to quit smoking, which would reduce their risk of lung cancer , 31. Secα-blocker usage. The potential interaction on the chemopreventive influence from combination usage of α-blocker and aspirin might exist. These associations appeared to attenuate with longer duration of usage. Due to the observational nature of this study, the results shall be interpreted carefully. More corroborations on these findings are needed to focus on the different settings in future studies.In conclusion, through the large population-based retrospective cohort study in Hong Kong, the lung cancer incidence risk from long-term anti-hypertensives treatment was assessed. Anti-hypertensive medication was associated with a lower risk of lung cancer, especially for male patients or those aged over 65 years old. Similar conclusions were reached when assessing the lung cancer risk from the only ACEi/ARB and Additional file 1.


Arabidopsis thaliana) have been published, there is still need for a de novo reference map for both root and especially above-ground cell types. As the plants’ transcriptome substantially changes throughout the day, shaped by the circadian clock, we performed scRNAseq on both Arabidopsis root and above-ground tissues at defined times of the day. For the root scRNAseq analysis, we used tissue-specific reporter lines grown on plates and harvested at the end of the day (ED). In addition, we submitted above-ground tissues from plants grown on soil at ED and end of the night to scRNAseq, which allowed us to identify common cell types/markers between root and shoot and uncover transcriptome changes to above-ground tissues depending on the time of the day. The dataset was also exploited beyond the traditional scRNAseq analysis to investigate non-annotated and di-cistronic transcripts. We experimentally confirmed the predicted presence of some of these transcripts and also addressed the potential function of a previously unidentified marker gene for dividing cells. In summary, this work provides insights into the spatial control of gene expression from nearly 70,000 cells of Arabidopsis for below- and whole above-ground tissue at single-cell resolution at defined time points.Although several large-scale single-cell RNA sequencing (scRNAseq) studies addressing the root of Arabidopsis  and above ground tissues (at the end of day and the end of night) revealed marker genes, cell type differences, and similarities. Arabidopsis thaliana) root tissues, but this approach masks information about cellular heterogeneity within a given tissue (Bulk collection of tissue-specific cell populations by fluorescence-activated cell sorting (FACS) has provided valuable insights of gene expression within, for example Arabidopsis  parts of Arabidopsis at clearly defined diel time points of harvest and growth conditions. Most available scRNAseq studies do not indicate an exact time point of harvest although it is well established that the transcriptome changes significantly depending on the circadian clock especially between the end of the day (ED) and the end of the night (EN). We address these aspects and provide insights into the spatial control of gene expression at single-cell resolution from nearly 70,000 cells of Arabidopsis for below- and, for the first time, whole above-ground tissues at ED and EN time points. This approach allows us to answer questions, such as, what common cell types and corresponding markers can be found between root and shoot, or how does the single-cell transcriptome change in certain tissues depending on the time of the diel cycle?To profile both Arabidopsis root and above-ground cells in single-cell resolution and to generate the necessary scRNAseq library, we customized the Drop-seq protocol  for planH2B-Venus fluorescent marker under tissue-specific promoters marking the phloem pole pericycle , the xylem pole pericycle , and post-meristem differentiated cells (line V311)  conditions (12/12-h photoperiod) on 0.5× MS plates supplied with 0.6% w/v sucrose. The used reporter lines were expressing the ne V311) . For eacne V311) , 75 min nd at EN  above thnd at EN . The cDNnd at EN  was usedACTIN2  verified successful RNA capture and cDNA production, and presence of SUCROSE-PROTON SYMPORTER 2 , a well-known phloem companion cell marker gene, confirmed protoplasting of vascular phloem cells in all three single-cell libraries. Therefore, we concluded that our scRNAseq libraries contain not only transcripts from the most abundant or easily protoplasted cells, but also transcripts from cells present in deeper layers and with a more rigid cell wall. Rare cell types like the root QC indicated by WUSCHEL RELATED HOMEOBOX 5    were notACT2, KNOTTED-LIKE FROM ARABIDOPSIS THALIANA , and SUC2 transcript presence as markers for successful cDNA production and the presence of companion cells, vascular cambium/meristem, and stomata cells. In addition, to confirm that the light treatment and time of harvest reflected ED and EN, two transcripts: LUX ARRHYTHMO , peaking at the ED, and LATE ELONGATED HYPOCOTYL , peaking around EN were used to validate that the libraries reflect the expected diurnal transcriptomic profiles , thus, we obtained a good reproducibility of the samples , correlation-based clustering, and differential gene expression analysis. The root and shoot libraries are separated among PC1 (32.93% of variance) and scRNAseq and reference RNAseq libraries are separated among PC2 (29.34% of variance) . Similar samples . Also, t2FC| > 1; Next, we tested for differential expression (DE) of genes between scRNAseq libraries and their corresponding reference RNAseq libraries  among the most significantly changed genes. This is also reflected by GO enrichment analysis, where “circadian rhythm”  and “response to light intensity”  are among the most enriched categories  and reference RNAseq libraries  with an overlap of 1,432 genes . As expetegories .To facilitate the identification of cell types, we used Seurat to separate the scRNAseq data into clusters for root and ED and EN shoot datasets. Prior to clustering, we filtered out the data of cells with too few reads, with large amounts of plastidial transcripts, or with unspecific gene-expression that are likely to be noise . For rooNote that while the information content per cell seems lower than in other published scRNAseq datasets, one must consider two things: Firstly, most studies use a different method, that is 10× Chromium which provides better read coverage per cell. Secondly, these numbers are highly cutoff-dependent. For example, by increasing the minimum number of transcripts/genes per cell in root from 500/200 to 2,000/1,000, the average number of transcripts/genes per cell rises from 1,994/822 to 4,386/2,040. However, instead of nearly 20,000 cells, only <3,000 cells with high depth would remain for clustering. As the main purpose of this study was the discovery of potentially rare cell-types and transcripts, we decided for a lower cutoff, which results in a larger number of analyzed cells and subsequently more clusters to be analyzed further.For root, the analysis resulted in 35 clusters, whereas for the individual ED and EN datasets, we obtained 37 and 25 clusters, respectively. We also combined the scRNAseq data from ED and EN to obtain a pooled ED/EN dataset, for which the number of clusters was 16. Finally, we confirmed that cells from the same replicates are well distributed across all clusters .In order to evaluate the robustness of each clustering, we randomly subsetted 80% of our library reads (100 times) and repeated the computational pipeline. We then checked what proportion of cells (0–100%) that previously formed a cluster are still co-occurring in a single cluster when using the subsetted reads for clustering . The aveNext, the most significantly enriched genes for each cluster were identified , in ordeFor this study, whole above-ground tissue harvested at two different time points (ED and EN) was submitted to scRNAseq. Thus, the transcriptome data represent a mixture of leaves of different developmental and physiological stages and shoot apices. The cluster annotation was first performed for the individual ED and EN and then for the combination of the ED and EN datasets.Unlike the Arabidopsis root tissues, different cell types of leaves at different developmental stages are less well understood. There is also a lack of high-resolution tissue-specific transcript data and of available transgenic marker lines. Because of this, the shoot-derived clusters were annotated in a broader fashion than the root-derived clusters.First, we separately clustered the cells from ED and EN samples . In summIn order to ascertain tissue-type/cluster identification, we pooled the datasets from both time points  by normaWe identified clusters representing the main tissues of the shoot , that isNotably, we do not find stomata as a unique cluster in our dataset, instead key transcripts known to specifically regulate stomatal development are found distributed across several clusters. We therefore concluded that the dominant photosynthesis expression pattern prevents these cells from clustering as a specific cell type.The MapMan functional category “Carbohydrate metabolism” provided a good example for ED and EN differential gene enrichment, although this category did not fall into the highly enriched ones. In the subcategory “starch metabolism biosynthesis” created for the pooled shoot ED+EN, a clear enrichment was found for the cluster 15 (and 1), representing mesophyll cells where most of the starch is synthesized . To furtNotably we see a change of cluster distribution in the clustering between ED and EN. For example, at ED 10 mesophyll clusters and at EN only 4 mesophyll clusters appear to be present . To gainSUCROSE PHOSPHATE SYNTHASE 4F  and SUCROSE PHOSPHATE SYNTHASE 2F  (CA(2+)/H(+) ANTIPORTER1  necessary for the assembly of the PSII core subunits and the oxygen-evolving complex  genes relative to the other ED mesophyll clusters. Sucrose production seemed to be high in clusters 4 and 7 marked by relative high expression of SUCROSE SYNTHASE 1  and SUS2 (AT5G49190) with the latter not detected in in clusters 2, 3, 6, 13, 24, and 27.Furthermore, the mesophyll clusters show distinct metabolic features in such that sucrose–phosphate synthase encoding transcripts of 5G11110)  was low  complex . AnotherTo further characterize circadian rhythm (ED or EN)-derived changes at the tissue level, we analyzed the datasets regarding complexity. Complexity is here defined as the variation in response, such that a tissue exhibiting a uniform response to a stimulus would be considered less complex than a tissue responding heterogeneously. For this analysis, the clustering of the shoot scRNAseq dataset was repeated without the previously described normalization used to create the pooled ED/EN dataset . For eacGRP7 that would have been undetected without looking separately at the clusters and time points.Importantly, this approach allowed us to investigate tissue-specific markers depending on the time of the day. In order to find ED- or EN-specific markers, we analyzed ED and EN cells separately for (i) clusters with a mixture of ED and EN cells and (ii) clusters with same annotation but split for ED and EN cells . For exaIn this study, protoplasts were made from approximately 2–3-cm long roots and, thus, included the root apical meristem (RAM) and mature regions of the root with different stages of lateral root initiation sites . The rooPLETHORA 2  transcripts (cluster 29)  (XPP cluster 13b)  transcript (procambium annotated clusters 11 and 19) which is a programmed cell death/autophagy marker specifically expressed in the cells surrounding the emerging lateral root   and GATAter 13b) , respectral root . Also, wral root .One interesting aspect was that among the different root cell types, the root cap (cluster 15) shows significant enrichment of several MapMan functional categories related to secondary metabolism  pinpointA closer look at those highly enriched MapMan categories unveiled specific tissue and/or time of the day gene responses which could be also considered a validation of the experimental procedure used. For example, the category “chromatin organization” was highly enriched in the root cluster 3, as well as in the shoot ED/EN cluster 4, comprising amplifying cells of RAM and SAM, respectively . Both chP-value: 7.22E−26). Here, the uncharacterized gene “AT5G16250” showed the highest FC (i.e. 10.001) in cluster 32 (root) and was more closely investigated (see below).We also investigated potential overlaps in root and shoot clusters by calculating the Jaccard index, that is, the ratio of intersection and union, for the markers of all cluster pairs . While mAT5G16250 transcript predicted to encode a transmembrane protein. Thus, to confirm its specific expression, we checked the AT5G16250 expression at different cell cycle stages using CycleBase 3.0 . To confirm the prediction that MERCY1 is a cell cycle marker involved in the regulation of the cell cycle, we first analyzed the expression of the gene in planta by RNA in situ hybridization on longitudinal sections through root tip and SAM of wild-type plants  and S-phase marker HISTONE H4  at the SAM of Col-0 wild-type and mercy1 mutant plants indicate that knockout of MERCY1 gene affects the progression of the cell cycle  show a growth phenotype (mercy1 mutant compared with Col-0 plants (mercy1 (P < 0.05) seedlings , or hyponasty during the vegetative growth phase. Although the final bolt height of mercy1 plants was substantially shorter compared with Col-0 plants (mercy1 flowered significantly earlier compared with Col-0 based on the RLN (P < 0.01) and DTF (P < 0.001)  . We alsoe plants .MERCY1 is involved in meristematic cell division in a regulatory capacity with pleiotropic effects on flowering time, bolting, and root formation.Taken together, while its exact role is unknown our results indicate that CHOLINE KINASE1  previously shown to be a graft-mobile transcript depending on the presence of the di-cistronic tRNA-like sequence (TLS) (CK1 (non-mobile) versus CK1-TLS (mobile) by accurately quantifying the two cistronic forms .We also addressed the presence of graft-mobile protein encoding transcripts  and theice (TLS) . To confCK1-TLS transcript compared with mono-cistronic CK1, in the procambium cluster 16a , PPP cluster 20b  and endodermis cluster 31  (CK1 transcripts were identified as CK1-TLS (CK1-TLS was found significantly enriched in mesophyll (cluster 0 for both ED and EN), in epidermis , and in senescent cells (cluster 8 for ED and cluster 18 for EN)  . On aver CK1-TLS , whereas CK1-TLS . Similar for EN) .CK1 and CK1-TLS in the cell types where the differences were predicted to be high, that is we selected clusters 17  and 31 . We used the endodermis and non-hair cell marker lines described in CK1 and CK1-TLS  and CK1-TLS (392 bp) in different ratios and at different concentrations  that were also markers for both root and shoot clusters  and EN/ED shoot  . Out of these, 9,228 transcripts were predicted as additional isoforms of the existing genes. The remaining 193 predicted transcripts were present in previously non-annotated regions covering 125 non-annotated genes . Based oclusters . From th and 9a) . ATNG-47function . To confynthesis . To conf primers . These canscript . In summr > 0.9) and a high correlation between scRNAseq libraries and corresponding reference RNAseq libraries (r approx. 0.8), which is comparable to recently published datasets  roots of 7-d-old seedlings at ED and for (ii) above-ground tissue of 5-week-old plants at two distinct times of the day (ED and EN). We produced reference RNAseq libraries from the respective tissue by using the same cDNA protocol and RT primer employed for the scRNAseq library production. We observed a high reproducibility across replicates  was experimentally confirmed regarding its role in growth and development. MERCY1 was identified as a cell-cycle-related marker transcript in both shoot and root based on its specific presence in clusters identified as dividing cells. It is well-established that plant growth and development depends on the interplay between meristematic cell division and post-meristem cell expansion  preferentially expressed in distinct cell types suggesting the potential importance of such loci in the Arabidopsis genome. Furthermore, we exploited the power of scRNAseq to go beyond the “traditional” scRNAseq analysis and look at the individual transcripts from the sequence level. Using this approach, we investigated the distribution of di-cistronic transcripts across the cell types and separately quantified the mono- and di-cistronic (CK1-TLS) versions of the CK1 transcript. We experimentally confirmed the enrichment of CK1-TLS within the endodermis via FACS and RT-qPCR. Beside the endodermis, the graft-mobile CK1-TLS  and demonstrate its role in meristematic growth. Furthermore, we identified non-annotated transcripts that seem to serve as tissue-specific markers and analyzed the differential presence of mono- and di-cistronic transcripts within tissues.In conclusion, we have created a high-resolution single-cell transcriptomic map of Arabidopsis root at ED and above-ground tissues at ED and EN and identified tissues-specific markers for both time points. We found that depending on the time of the day, single-cell transcriptome changes occur in distinct tissues to variant degree. Interestingly, we observed that the most similar tissue type between root and above-ground tissue is dividing cells. This observation led us to investigate a previously uncharacterized marker of that cluster  estradiol-inducible fluorescent marker lines  with characterized expression patterns were used to produce scRNAseq libraries of the root using the Drop-seq protocol. Lines V161 and V171 express H2B-Venus in the PPP and the XPP under the promoter of AT5G09760 and AT3G29635, respectively. Line V311 expresses H2B-Venus in differentiated cells under the promoter of TCP DOMAIN PROTEIN 7  . Each line comprises an independent experiment and were considered replicates  . The plaplicates .www.stender.de) mixed with vermiculite (1:1) soaked with tap water, supplemented with boron (1.8 mg L−1) and the fungicide Previcur  and germinated in 12-h light/dark photoperiod (NDs) at 20°C in controlled environmental chambers (160 μE m−2 s−1). After 1 week, the seedlings were transferred to an 8-/16-h light/dark photoperiod  at 20°C/16°C for 1 week. Then plants were transferred into individual pots (five plants per pot) and grown under ND (12-h light/12-h dark) photoperiod with 22°C/18°C temperature . Four weeks after transfer, all above-ground tissue was used for protoplast preparation (n = 40 plants) and submitted to scRNAseq. Each of the three library pairs ED1/EN1, ED2/EN2, and ED3/EN3 was harvested within 24 h from the same batch of plants  and above-ground tissue from soil grown plants (20 plants) harvested at the same time points as for the scRNAseq libraries and, to avoid a potential preparation bias introduced by PCR amplification/tagmentation, the reference tissue cDNA libraries were prepared in the same way as the scRNAseq cDNA libraries . At 7 DAG, the seedlings were transferred to fresh 10% w/v agar 0.5× MS 0.6% w/v sucrose plates  using poly-T primers and used for RT-qPCR.For details, see https://github.com/broadinstitute/Drop-seq/). The final alignment files were used for quantifying expression of all genes using htseq (version 0.12.4). The raw counts were further normalized using DESeq2  of 0.05 and |logdatabase . We downdatabase  and perfP-value < 0.05) . A gene pair was considered to encode a di-cistronic transcript if it was supported by ≥100 reads. These gene pairs were further filtered to retain pairs if both genes are protein-coding and present on the same strand; or at least one gene is non-coding.CK1 (CK1-mono) and di-cistronic CK1 (CK1-TLS) were quantified from the single-cell RNAseq data with custom-made scripts. A transcript was annotated as CK1-mono if no reads from transcript overlap the tRNA region (AT1G71700) and the reads end at 1 bp before the start of the tRNA, and a transcript was annotated as CK1-TLS if at least one read from the transcript aligned to the tRNA region. The dot plots were plotted using ggplot2 package in R. Clusters were considered to be enriched in CK1-TLS if CK1-TLS was identified as a marker for the cluster  and CK1-mono was not.The mono-cistronic The sequencing data sets are available at the NCBI Sequencing Read Archive (SRA), BioProject ID PRJNA742744.Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: MERCY1 (AT5G16250) and CK1 (AT1G71697). Outline of the experimental workflow. RT-PCR and normalized expression of marker genes in the single-cell and reference tissues of root and above-ground samples. PCA, clustering, and correlations. DEGs affected by scRNAseq compared with published data sets. Single-cell transcriptome of root and above-ground samples prior filtering. t-SNE projection with cell colors for replicates. Analysis of cluster robustness. Enrichment of cluster marker genes in the MapMan functional categories. Above-ground cluster (pooled ED/EN) enrichment for MapMan categories related to starch metabolism. Enrichment of MapMan level 1 categories in above-ground samples. Marker genes in above-ground clusters. Root cap enriched MapMan categories related to secondary metabolism. MapMan category “chromatin organization” enriched in root and above-ground clusters related to RAM and SAM, respectively. Overlap of markers between root and above-ground (pooled ED/EN) clusters. Characterization of molecular function of AT5G16250 (MERCY1). Analyzing growth behavior of mercy1. Enrichment of graft-mobile annotated transcripts and di-cistronic transcripts in root and shoot (pooled ED/EN) clusters. Dot plots representing transcript accumulation of CK1, mono-cistronic CK1 and CK1-TLS transcripts for each cluster in above-ground clusterings. RT-qPCR of standard curves created using purified PCR products of CK1 and CK1-TLS. FACS of tissue-specific protoplasts and RT-qPCR of isolated cDNA.. List of primer sequences used. Summary of bulk and single-cell libraries used in the study. List of all predicted non-annotated transcripts and loci. List of differentially expressed genes. GO enrichment analysis of DEGs between ED and EN. Summary of clusters for root samples. Summary of clusters for end of day samples. Summary of clusters for end of night samples. Summary of clusters for pooled end of day + end of night samples. List of markers identified for root clusters. List of markers identified for end of day clusters. List of markers identified for end of night clusters. List of markers identified for pooled end of day + end of night clusters. Comparison of individual end of day and end of night clusters with pooled end of day + end of night clusters. List of markers identified for pooled end of day + end of night clusters without normalization. Comparison of pooled end of day + end of night clusters with and without normalization. List of markers identified between specific clusters for pooled end of day + end of night clusters without normalization. List of all predicted di-cistronic transcript pairs. Summary of CK1 and CK1-TLS transcripts identified in the root clusters. Maximum percentage of cells for the dot plots for all clusters. Protoplast isolation. ScRNAseq with Drop-seq. Reference tissue RNAseq. Quality control PCRs. Clustering and marker identification for single-cell samples. RNA in situ hybridization. Growth and morphology analysis. RT-qPCR of CK1/CK1-TLS and non-annotated transcript. Annotation of Arabidopsis reference genome and alignment.. MapMan annotation of protoplast-induced genes.. ED cluster annotation. EN cluster annotation. ED/EN cluster annotation.. Root cluster annotation.. Root cap and secondary metabolism.kiab537_Supplementary_DataClick here for additional data file.


Intercellular adhesion molecule-1 (ICAM-1) mediates extravasation of leukocytes, releasing proinflammatory cytokines or endogenous opioids in the inflamed tissue. Thus, ICAM-1 is a crucial component of peripheral antinociception. Previously, we demonstrated a significant correlation between the soluble form of ICAM (sICAM-1) in serum and pain intensity reported by chronic pain patients. The present study examines the role and kinetics of sICAM-1 in experimentally induced acute pain. Three groups of 10 subjects were exposed to 10 min of high (capsaicin-enhanced) or low-intensity heat pain or cold pain, respectively. Thermal stimuli were induced using a device for quantitative sensory testing. Topical capsaicin significantly increased heat pain intensity without the risk of thermal tissue damage. Pain intensity was recorded every minute during testing. sICAM-1 concentrations in serum were determined by ELISA before, immediately after, and 60 min after test termination. Among all experimental groups, sICAM-1 significantly decreased immediately after pain induction. After 60 min, sICAM-1 concentrations returned towards initial values. Interestingly, a linear correlation was found between the extent of sICAM-1 changes and the initial concentrations. Whereas high initial values led to a distinct decrease of sICAM-1, low concentrations tended to increase. There was no statistically significant correlation between levels or alterations of serum sICAM-1 and pain intensity reported by the test subjects. In contrast to our previous findings in chronic pain patients, the present results show that sICAM-1 values do not correlate with the intensity of acute experimental pain. However, we were able to detect short-term changes of sICAM-1 after induction of nociceptive thermal stimuli, suggesting that this marker is part of a demand-oriented homeostatically controlled system.  Pain is an experience comprising sensorimotor, mental and affective components. Despite the complexity of pain, the rated pain intensity is always assessed in daily clinical routine in order to adjust treatment. This is particularly true for acute, i.e., postsurgical pain. However, the assessment of pain may pose considerable problems for health professionals under certain conditions such as dementia. Since biomarkers for the quantification of pain do not (yet) exist, the type and extent of analgesic therapy is still determined by feedback and information given by the patients. Frequently used survey methods are scales developed to verbally, visually, or numerically record the intensity of perceived pain ,2. ThougIn the search for potential biomarkers to quantify pain intensity, some cytokines e.g., IL-7, IL-13), free fatty acids, or hormones  could be identified whose serum concentrations showed alterations in the application of experimental pain models ,6. Howev, IL-13, We found a significant correlation between both the initial values of sICAM-1 serum concentration and pain level as well as their respective changes . An association between sICAM-1 and pain has been demonstrated in both animal models and patients with other types of pain  ,13.In the present study, we therefore focused on the role and kinetics of sICAM-1 in acute experimental pain in healthy volunteers.ICAM-1, also known as CD54 (cluster of differentiation 54), is a cell surface glycoprotein, which is typically expressed on endothelial cells and cells of the immune system  . ICAM-1 Soluble ICAM-1 can be generated either by proteolytic cleavage of the extracellular domain of membrane-bound ICAM-1 by human leucocyte elastase, cathepsin G, matrix metalloproteinase-1 and -9 and TACE/ADAM-17, or by alternative splicing ,20,21,22The present study was designed to investigate the role and kinetics of serum sICAM-1 in acute pain. Therefore, thermal nociceptive stimuli were induced using a device for quantitative sensory testing. It was examined whether the absolute concentrations and/or the extent of alteration of the initial concentrations correlated with the reported pain intensity.Since ICAM-1 plays a critical role in inflammatory processes of any kind, it was essential to conduct the study without the most significant possible bias. Participants with possible confounding factors such as psychiatric diagnosis, acute or chronic physical diseases and current medication were excluded in advance. To induce experimental pain in healthy subjects, we first established an appropriate acute pain model . By usinLow-intensity heat pain model with application of a placebo cream High-intensity heat pain model with application of a capsaicin cream Cold pain model with application of a placebo cream The acute pain model included three experimental groups :Low-intInduction of a significant pain stimulus with an average perceived pain intensity of ≥6 on a numerical rating scale in the high-intensity heat pain and cold pain model over a test duration of 10 minHigh tolerance of the pain model by the study participants Avoidance of severe or irreversible tissue damage Moreover, the model was characterized by the following features:The pain models were programmed to maintain a temperature range of 0–50 °C and switch off automatically after 5 min. The test subjects were able to interrupt the program at any time in case of pain intolerance utilizing a remote control. The perceived pain intensity was recorded right before the test, every minute during the experiment, and finally 60 min after the first survey via a numeric rating scale. The test areas on both volar forearms  were prepared according to the randomised assignment. First, the skin was cleaned with an alcohol-based disinfectant. A hazelnut-sized amount of a commercial moisturiser (cold pain group and low-intensity heat pain group) or capsaicin cream  was then administered to an area of about 4 × 4 cm. An occlusive dressing was applied to enhance the effect. After 20 min, the dressings and cream residues were removed. The thermode was first attached to the right arm. After running through the assigned pain program, the thermode was changed to the contralateral arm to avoid thermal skin damage. In order to ensure the greatest possible exclusion of factors influencing pain perception, the experiments were always carried out between 4 and 7 p.m. at an ambient temperature of 21–23 °C (degrees Celsius). The experiments were always carried out by the same male investigator. The low-intensity heat pain group underwent the same program as the high-intensity heat pain model but instead of a capsaicin cream, a commercial moisturizing cream (placebo cream) was applied to the test area. The previous topical application of capsaicin in the heat pain group resulted in the same heat stimulus being perceived as significantly more painful. This setup was designed to detect whether changes in sICAM-1 serum levels are due to the application of the chemical agent capsaicin and if the extent of the perceived pain intensity correlates with absolute concentrations or concentration changes of sICAM-1 .The high-intensity heat pain model started at a thermode temperature of 32 °C (baseline). The temperature increased at a rate of 10 °C/s up to the first heat stimulus of 38 °C. The stimulus duration was 20 s each. After returning to the baseline temperature for a period of 0.5 s the next stimulus induction started. The temperature of the thermode increased stepwise by 1 °C after each heat impulse up to the maximum temperature of 50 °C. The program ended after five minutes. To prolong the pain induction without the risk of severe tissue damage, the program was restarted on the contralateral arm of the test subject . The cold pain model also started with a thermode temperature of 32 °C. The temperature decreased at a rate of 10 °C/s to the first cold stimulus of 2 °C. The stimulus duration was 40 s. Subsequently, the thermode temperature returned to 15 °C (baseline) for 4 s before a new stimulus induction was started. The temperature of the thermode was lowered stepwise by 0.5 °C after each cold impulse to the minimum temperature of 0 °C. After five minutes, the program switched off and another test run was performed on the contralateral arm . For the experimental study, healthy, adult, pain-free test subjects were recruited. The experiment involved 15 women  and 15 men . Exclusion factors were pregnancy and any type of acute or chronic physical or psychological disease. One subject had to be excluded afterward due to pathological hypaesthesia.The experimental participants were randomly assigned to three experimental groups of 10 subjects each. There was no cross-over-design. To achieve double-blinding, the preparation of the subjects and anonymization of the pain programs were carried out by an assistant investigator. Before the start of the trial, a standardized questionnaire was used to collect data from each subject regarding age, gender, education, weight, height, current and past illnesses, and medication intake over the last four weeks. The individual stress level was assessed using the German version of the Questionnaire for Actual Demands (KAB: Kurzfragebogen zur aktuellen Beanspruchung). By means of a 6-point scale ranging from 1 to 6 based on normalized adjectives , the KAB score was developed for assessing acute or chronic distress. Higher values indicate increased levels of distress . Cut offg for 15 min. The blood plasma was then pipetted into plastic aliquots tubes (specimen á 150 µL) and immediately frozen at −80 °C. For the quantitative determination of sICAM-1 concentrations a Human ICAM-1/CD54 Allele-specific immunoassay  was used. Quality check and double measurements were performed. Before, at the end and one hour after starting the experiment a sterile, a venous blood sample was taken from each test subject from the median cubital vein (EDTA sample tubes). Since it is unclear whether an inserted venous catheter might influence the serum concentration of sICAM-1, blood samples were taken by single punctures from alternating sides. Within 30 min the blood samples were centrifuged at 1000× SPSS Version 25 of IBM Statistics was used for statistical analysis and randomization of the experimental groups. The data were tested for normal distribution using the Kolmogorov–Smirnov test. Differences in plasma sICAM-1 concentrations were tested for significance using the t-test for paired samples. Correlation coefficients were determined according to Pearson. Whether the mean values of the collected data differ depending on the experimental group was determined by univariate analysis of variance with repeated measurements and post hoc test (LSD). An a priori sample size calculation was performed with G-Power. We performed a power analysis based on our data of chronic pain patients with an observed effect size of 6.68. Since the standard deviation of the differences in chronic pain patients varied depending on the perceived pain level, we chose the largest observed standard deviation as the basis for calculation for the final case number estimate . p < 0.001), but not within the high-intensity pain groups (p = 0.64). The experimental design described above allowed the induction of a significant heat and cold pain stimulus with an average perceived pain intensity of NRS ≥ 6. In the low-intensity heat pain group, the average pain intensity over a 10-min test duration was NRS = 1 see . There wp = 0.003)  caused a significant alteration of sICAM-1 concentrations across all test participants. Mean sICAM-1 concentration before the start of the experiment was 204.89 ng/mL (SD = 35.46). Immediately after induction of pain, mean sICAM-1 was 197.5 (SD = 27.13). At 60 min after starting the experiment, mean sICAM-1 value was 202.79 ng/mL (SD = 29.76). The difference in mean sICAM-1 concentration before and immediately after induction of pain was statistically significant (003) see . To test whether the alteration in sICAM-1 concentration depends on the type of pain (heat or cold) or pain intensity (high-intensity or low-intensity), three different statistical analyses were performed.p = 0.037). At 60 min after starting the experiment mean value of sICAM-1 concentrations nearly approached the baseline with 204.67 ng/mL (SD = 23.43). In the low-intensity heat pain group, no statistically significant correlation between the pain intensities perceived and the serum concentrations of sICAM-1 or changes in these concentrations could be found. . Immediately after the end of heat induction, mean sICAM-1 value was 200.08 ng/mL (SD = 22.11). The difference in sICAM-1 concentration before and immediately after heat induction was statistically significant (und. see  p = 0.176). After the trial, mean sICAM-1 concentration in serum approached the baseline value. The mean value of the sICAM-1 concentrations 60 min after starting the experiment was 193.04 ng/mL (SD = 19.51).In the high-intensity (capsaicin-enhanced) heat pain group, mean sICAM-1 value before starting the experiment was 194.33 ng/mL (SD = 23.79). Immediately after heat induction, mean sICAM-1 value was 189.47 ng/mL (SD = 18.61). The difference in sICAM-1 concentration before and immediately after the pain induction was not statistically significant . After the trial, mean sICAM-1 concentration in serum approached the baseline value. The mean sICAM-1 concentration 60 min after starting the experiment was 211.53 ng/mL (SD = 42.94). In the cold pain group, mean sICAM-1 value before starting the experiment was 212.69 ng/mL (SD = 51.08). Immediately after the end of pain induction, mean sICAM-1 value was 203.68 ng/mL (SD = 38.81). The difference in sICAM-1 concentration before and immediately after pain induction was not statistically significant . Low initial concentrations even led to an increase of sICAM-1 in serum after running through the experiment  of pain is responsible for the obtained results. However, it cannot be excluded that this observation might also be related to the large standard deviation of the initial sICAM-1 values in the healthy subjects. In contrast to our previous findings concerning chronic pain patients, there was no correlation between sICAM-1 concentration and pain intensity in the present study, indicating that measurement of this marker is not suitable for the quantification of acute pain. The observed differences in the role of sICAM-1 in acute and chronic pain may be due to different pathophysiological mechanisms underlying the two entities. In particular, dysfunction of the hypothalamic-pituitary-adrenal (HPA) axis often described in chronic pain patients should be considered because of its extensive influence in the expression and function of immune cells and adhesion molecules. Immunocompetent cells  exhibit receptors for neuroendocrine products of the HPA axis . The imbalance of the HPA axis and its neurotransmitters and resulting long-term alterations, such as downregulation or increased resistance of targeted receptors , lead to changes in trafficking adhesion, proliferation and cytokine secretion of immune cells ,34,35,36In the experimental setup described here, high initial values of sICAM-1 distinctly decreased, whereas low concentrations tended to increase due to pain induction. In both cases, an approximation towards initial values was seen at the 60-min time point. The increase or decrease of sICAM-1 serum concentrations and the extent of alteration depended on the baseline concentrations. Since the soluble form of ICAM-1 is also a ligand of LFA-1-expressing leukocytes, the observed attenuation might be explained by receptor-binding, followed by an increase through proteolytic cleavage of upregulated membrane-bound ICAM-1, alternative splicing or dissolving from any receptor targeted by sICAM-1. The mechanism of response regulation takes place with a latency. However, according to this theory, an initial decrease in sICAM-1 would be expected among all experimental subjects. Considering the linear relationship between baseline and extent of alteration of sICAM-1 concentration, the changes seem to be demand-oriented as a possible expression of immunological homeostasis. This mechanism could allow an adequate immune reaction to be triggered or an overshooting response to be prevented. The results obtained in this study suggest a balanced and rapidly adaptable system of circulating immunomodulatory proteins, including sICAM-1. They also provide information about the short-term kinetics of sICAM-1 after experimental pain induction for the first time. The regulating mechanism responsible for the changes in sICAM-1 concentrations described here needs to be investigated in future studies.The fact that the abovementioned results could be obtained in all experimental groups reveals an important limitation of the present study design: it cannot be conclusively answered whether the thermal stimulus, the skin penetration caused by blood sampling, or even simply the induction of acute stress led to the changes of sICAM-1 concentrations. It has to be taken into account that pain is a complex experience, including physical, psychological, and social aspects. This is particularly true for chronic pain, which the “bio-psycho-social pain model” commonly defines. However, psychological stress is also an essential part of acute nociception. Therefore, it can never be ruled out that changes observed in acute and chronic pain are stress-induced. In fact, it has been shown that stress and mental distress influence the expression of adhesion molecules. Heinz et al. observed an increase of sICAM-1 concentration two hours after the induction of acute psychological stress followed by normalization on the following day . TherefoIn further studies providing a more extended observation period, it will be of interest to evaluate whether the nociceptive stimuli, in contrast to, e.g., acute mental stress, cause a long-term upregulation of membrane-bound and soluble ICAM-1 as a possible sign of increased peripheral antinociception. In conclusion, we found that in contrast to our previous results in chronic pain, sICAM-1 levels do not correlate with the intensity of acute experimental pain. However, we were able to demonstrate the short-term kinetics of this adhesion molecule after induction of painful stimuli in healthy volunteers for the first time. Our data suggest that sICAM-1 is part of a demand-oriented homeostatically controlled system involved in the experience of pain.


Background and objectivesIn comparison to real-time polymerase chain reaction (RT-PCR) testing, blood-related parameters including absolute lymphocyte count (ALC) and neutrophil-to-lymphocyte ratio (NLR) carry an indeterminate potential in the assessment of corona virus disease 2019 (COVID-19). Our main objective was to assess their efficacy in timely identification of COVID-19 patients and to determine whether these biomarkers can be employed as an early diagnostic tool in patients presenting with symptoms suggestive of COVID-19.Methodology3 cells/mm3 was considered as the cut-off value. Statistical analysis was conducted via SPSS 23.0 . Chi-square and independent t-tests were used to correlate various data variables, while p-value <0.05 was considered significant.This cross-sectional study was conducted at the Emergency Department of a Tertiary Care Hospital in Rawalpindi, Pakistan from November 2020 to March 2021. Patients suspected to have COVID-19 on a clinical basis  were selected by using convenience non-probability sampling. RT-PCR was used to diagnose COVID-19 after evaluating NLR and ALC of the sample population. An NLR = 3.5 and ALC < 1 x 10Results3/mm3, significantly lower than RT-PCR negative cases (p < 0.001). In addition, NLR was drastically elevated for RT-PCR-positive individuals (p < 0.001) while it also had a distinctly high specificity of 91.7% among COVID-19 patients. Additionally, NLR did not correlate with any of the baseline patient-related parameters .Out of the 172 subjects included in the study, the mean age was 40.6 ± 10.0 years, while 51% of individuals were males. Fever was found to be the most prevalent complaint (94%). Double RT-PCR testing showed that 51.2% of the population was RT-PCR positive, having a mean ALC of 1.4 ± 0.9 x 10ConclusionNLR and ALC are potentially efficacious measures for an early diagnosis of COVID-19, and can be possibly utilized for an early diagnosis of COVID-19 suspects. Corona virus disease COVID-19) has stringently challenged the fortitude of healthcare facilities throughout the globe, and has equivalently affected all walks of life. Ever since the pandemic began in late 2019, polymerase chain reaction (PCR) testing kits have been made available to diagnose the disease among patients suspected of clinical manifestations related to COVID-19. Ever since the emergence of COVID-19, real-time polymerase chain reaction (RT-PCR) has been widely implemented to allow a preliminary detection of COVID-19, and to help isolate RT-PCR positive individuals until a “double negative” PCR is confirmed [9 has strAt present, COVID-19 real-time PCR is considered as the first-line laboratory test for testing the clinically suspected population. In comparison to the high-resolution computed tomography (HRCT) scan, RT-PCR offers a sensitivity as low as 63%  and as hTo date, only a few credible studies have been conducted to ascertain whether NLR and absolute lymphocyte count (ALC) can be accurately used as an early predictor of RT-PCR positivity among COVID-19 suspects -15. FurtObjectiveThe objective of this study was to assess the effectiveness of CBC-based tools, i.e., NLR and absolute lymphocyte count (ALC) in the early detection of COVID-19 RT-PCR positive individuals.Study design and sampling techniqueThis study was conducted as a cross-sectional analysis at the COVID-19 Emergency Department of a Tertiary Care Hospital in Rawalpindi (Pakistan). The study lasted for a total duration of five months from November 2020 to March 2021. The participants were selected on the basis of non-probability convenience sampling technique. The sample population comprised a total of 255 patients, all of whom had clinical manifestations suggestive of COVID-19 .Inclusion and exclusion criteriaThe study population was selected only if they fulfilled all of the following criteria: (1) Any of the above clinical manifestations indicative of COVID-19; (2) Duration of illness ≤ 2 days and (3) Patient age ranging from 25 to 60 years (to ensure a satisfactory infectivity rate) . On the Operational definitionsClinically suspected individuals  were marked as “possible” cases of COVID-19.3 cells/mm3  was considered as a major indicator of COVID-19.An optimum range of NLR has been estimated to be 0.78-3.53 (Mean ± SD = 1.65 ± 1.96) . In thisA patient was characterized as “COVID Positive” only if they turned out a minimum of two positive Serial RT-PCR Tests.Data collection and analysisAn informed consent was obtained from all the participants while an ethical permission was also granted from the hospital ethical committee. The demographic details, a brief history of COVID-19 related symptoms, any history of contact with COVID-19 patients or with any potential carriers of infection, and CBC parameters including the neutrophil count, ALC and NLR were entered into a pre-designed proforma. Samples for RT-PCR were obtained via nasopharyngeal swabs, and examined at the Emergency Department laboratory of the respective hospital in Rawalpindi.The statistical parameters were analyzed by means of IBM SPSS version 23.0 . Pearson’s chi-square test and independent t-test were used to assess the statistical correlation of data. Odds ratio was calculated for both ALC and NLR values. A p-value < 0.05 was considered significant. Moreover, sensitivity and specificity values were evaluated for NLR and ALC measures by using the standard formulae.All the statistical data were obtained from a sample of 172 “possible” COVID-19 patients. Mean age was calculated to be 40.6 ± 10.0 years while approximately 51% of patients were males. More than 90% of the participants presented with fever, whereas cough was persistently present in up to 73% of the subjects. Shortness of breath was the next most common presenting complaint inflicting ~24% of individuals. Furthermore, a contact history was significant for less than 1/3rd of the study population , as evaluated through unpaired t-test analysis.Double RT-PCR testing confirmed COVID-19 in 51.2% of individuals of the sample population. Mean CBC values were computed for all of the participants. Mean TLC was 8.4 ± 3.4 x 10The mean NLR did not show any substantial variation with respect to the baseline features of the patients including their age, gender, and presenting clinical manifestations Table . AlthougIn terms of establishing an early diagnosis of COVID-19, NLR had a remarkably high specificity of 91.7% whereas this CBC measure had only a moderately high sensitivity (60.2%). For ALC, specificity was 96.4% whereas sensitivity stood quite low at 27.3%.This study has quantitatively measured the efficacy of hematological testing in predicting the probability of positive COVID-19 RT-PCR results. The authors have statistically elaborated that both ALC and NLR are fairly valuable tools in the early detection of corona virus, and that none of the baseline parameters are correlated with the variation in NLR. Having a remarkably high specificity, both ALC and NLR are useful measures for ruling out a diagnosis of COVID-19 among the clinical suspects of the infection. These results hold significance for directing the referral of these potential COVID-19 cases for an immediate RT-PCR test. Furthermore, NLR and ALC hold substantial relevance as comparatively cheaper as well as readily available diagnostic tools for COVID-19.Although there has not been much progress in the evaluation of NLR or ALC as a screening tool for COVID-19, still a number of authors have independently declared these two parameters as viable instruments for predicting the overall prognosis of patients. In contrast to moderately ill COVID-19 patients, Liu et al. have found that severely ill cases possess a mean NLR which is approximately four times higher than the former . MoreoveViral infections, in general, have often been proposed to induce secondary lymphopenia among the host organisms. Lymphopenia has been attributed to the proinflammatory state of body during the course of COVID-19 infection. A systemic cytokine storm is characterized by markedly raised levels of interleukins , and tumor necrosis factor-alpha (TNF-α), which contribute to the pathogenesis of lymphocyte apoptosis. Furthermore, it has been hypothesized that the virus exerts its cytotoxicity directly on the lymphocytes, which is mediated through angiotensin-converting enzyme-2 (ACE-2) receptors expressed on cellular membranes of the latter . In addi3/mm3 at the time of their emergency admission were predominantly at high risk of facing an early mortality [Low absolute lymphocytic counts have been implicated in COVID-19 by several authors. In one study, lymphopenia was a consistent laboratory finding encountered within the blood of up to 82% of deceased COVID-19 cases . A retroortality . Contrarortality .Only a handful of authors have hypothesized the applicability of ALC as an early diagnostic marker for COVID-19. One meta-analysis showed that ALC was the single most efficient laboratory assessment tool in emergency scenarios where more than 50% of patients had a subnormal lymphocyte count . In anotLimitationsAlthough the sample size was statistically calculated for this study, one of its major drawbacks is the relatively limited sample population which was confined to only a single medical facility. This could have potentially influenced the statistical significance of our results. Additionally, different RT-PCR kits show remarkable differences in terms of their relative sensitivity and specificity, and these observations are quite well-documented , 32. ThiAn increased neutrophil-to-lymphocyte ratio, as well as lymphopenia, are substantially effective tools in the early detection of COVID-19. With larger trials, it might become possible to consistently use these laboratory measures prior to referring patients for COVID-19 RT-PCR testing. Furthermore, a significantly high specificity for NLR has the potential to allow its application for ruling out the probability of a positive RT-PCR result. Our study can significantly impact the care of COVID patients in resource-poor settings where it can be utilized to triage patients most in need of a PCR test for the diagnosis of COVID-19 infection.


Child well-being concerns amidst the COVID-19 pandemic have been reported from countries with strict lockdowns and school closures. Sweden's middle school students attended school as normal during the pandemic, but it is still unknown how their well-being has changed during the pandemic. This study aimed to assess differences in Swedish students' psychosocial well-being from before to during the pandemic. Longitudinal data (N = 849) were collected via self-report surveys across two time-points separated by approximately one year. The second data collection took place 8–9 months after the start of the pandemic in Sweden. We measured psychological adjustment, relationships to significant others and school adjustment. Findings revealed significant mean-level decreases in students' school adjustment during the pandemic. There were no meaningful mean-level decreases in students' relationships to significant others. Students' psychological adjustment showed significant but mostly negligible mean-level decreases, and no differences in emotional problems during the pandemic. The findings are explained based on students' developmental stage and the handling of the pandemic in the Swedish school context. Based on this first longitudinal study on students' wellbeing during the COVID-19 pandemic, we conclude that Swedish middle school students who continued formal schooling show mainly positive adaptations, and thus appear to be resilient during the COVID-19 pandemic. •This is the first longitudinal study focusing on Swedish middle school students' psychosocial well-being during the COVID-19 pandemic.•Significant mean-level decreases were found in students' school adjustment, quality of relationships to significant others and psychological adjustment, when comparing levels from prior to the pandemic to during the pandemic.•Findings are explained based on students' developmental context and the context of the COVID-19 pandemic in Sweden•Despite significant decreases in students' psychosocial well-being, students showed no differences in emotional problems, showing support for students' resilience during the COVID-19 pandemic. Even though longitudinal research is needed to assess the impact of the pandemic, only a small minority of studies have been longitudinal with pre- and mid-/post-pandemic comparisons of children's well-being. RQ1Is there a difference between Swedish students' psychological adjustment prior to and during the COVID-19 pandemic?RQ2Is there a difference between Swedish students' quality of their connections to significant others prior to and during the COVID-19 pandemic?RQ3Is there a difference between Swedish students' school adjustment prior to and during the COVID-19 pandemic?In sharp contrast to many other societies, Sweden has allowed schools up to grade six to remain open and continue formal schooling during the pandemic. Recent longitudinal findings from Sweden have found that adolescents – for whom schools have been partly closed – have experienced slight decreases in their psychosocial well-being during the first year of the pandemic . Still, 2We analyzed data from children in 30 middle schools in western Sweden belonging to the longitudinal Peer Relations In School from an Ecological perspective (PRISE) project . Data we2.1SD = .03, range 9–11 years) and 11 years at T2 . Of participants, 403 were boys, 440 girls, and 6 gender non-conforming. Overall, 93.5% were of Swedish ethnicity (determined by asking participants where they were born), 88.6% of parents were from Sweden or another Scandinavian country, while 11% of both parents were born in another country. To assess participants’ socio-economic status (SES), students were asked whether they had their own room, with 86.4% reported that they had their own room at T1 and 89.5% had their own room at T2.Mean age at T1 was 10 years (2.22.2.12.2.1.1none of the time to (6) all of the time .The Children's Hope Scale CHS;  had six 2.2.1.2The Children's Self-Efficacy Scale (CSES) had four items relating to participants' ability to be assertive and expressive, rated on a scale from 0 to 100 how certain they were with their capabilities to, for example, “Stand up for myself when I feel I am being treated unfairly” and “Express my opinions when other classmates disagree with me” . The measure was taken from 2.2.1.3not true at all to (5) completely true.The Single-Item Self-Esteem Scale SISE;  had one 2.2.1.4not true, (2) partly true, and (3) completely true .The subscale of emotional problems from the Strengths and Difficulties Questionnaire SDQ-sve,  had five2.2.22.2.2.1never to (6) always. An example for perceived social support from parents was, “My parents … Listen to me when I need to talk” . An example for perceived social support from close friends was, “The friends I am with the most … Understand my feelings” .The subscales of perceived social support from parents and close friends from the Child and Adolescent Social Support Scale CASSS;  had thre2.2.32.2.3.1The subscale of perceived social support from teachers from CASSS  had five2.2.3.2never to (6) always .We asked participants two questions about their well-being in school, “I enjoy being in school” and “I feel safe at school”. The questions were answered on a 6-point Likert scale, ranging from (1) 2.2.3.3We asked participants four questions about their well-being in class, examples are, “In my class we help one another” and “In my class we are kind to one another”  , X2 = 1471.89, df = 1477, p = .532. An index variable was made for each psychosocial factor for paired sample t-tests. Visual inspections of variable distributions, as well as Shapiro-Wilk tests, indicated violations of the assumption of normality. Despite the non-normality of the data, we chose to use parametric paired-sample t-tests, as there is evidence that most parametric tests are robust enough to withstand non-normal distributions , class and school well-being , and students' self-esteem (M difference = -.31). There were no significant differences in students' emotional problems from T1 to T2. Overall, mean-level decreases were present in students' psychosocial well-being during the COVID-19 pandemic. The largest mean-level differences were found in students' school adjustment, in perceived support from teachers, school and class well-being, and in students' psychological adjustment in their self-esteem. These differences, however, had small effect sizes. Notably in students' psychological adjustments, students showed no differences in their emotional problems, and negligible differences in their sense of hope and self-efficacy.Descriptive statistics for the study variables and the paired sample ohen's d  standard4In this study, we aimed – for the first time known – to find out whether there were differences between middle school students' psychosocial well-being prior to and during the COVID-19 pandemic whilst attending school in Sweden. We compared students’ psychosocial factors of school adjustment, quality of connections to significant others and psychological adjustment prior to the pandemic (T1) to during the pandemic (T2). In sum, 8–10 months into the pandemic, students had slightly lower self-esteem compared to before the pandemic, felt slightly less supported by their teachers, and felt slightly less well at their schools and classrooms, but showed no differences in their experiences of emotional problems.These findings need be understood in relation to the context of students' developmental stage and the COVID-19 pandemic. Middle school students in Sweden are in the developmental stage of late childhood/preadolescence. This is a sensitive stage, where many changes, e.g., hormonal, emotional, cognitive, and social occur . TypicalIt is possible that the notable changes in students' school environment could be linked to the differences found in student's self-esteem. The absence of differences in emotional problems contrasts most international literature. This discrepancy may be due to international policy differences in approaches to the COVID-19 pandemic. Most countries that reported poorer mental well-being amongst children and adolescents, had strict lockdowns, curfews, and school closures. Finally, despite concerns about children's relationships with parents and close friends due to stress and social distancing during the pandemic , SwedishThis study gives support to prior findings that suggest children's well-being return to similar pre-pandemic levels as time goes on . We argu4.1The foremost limitation of this study is that we were not able to directly measure the participants' perception and experience of the COVID-19 pandemic. We cannot determine direct effects of the pandemic on psychosocial well-being. Presumably, this pandemic has changed everyone's day-to-day lives. Furthermore, we relate the study findings to students' developmental context, and by that we can infer what may be driving some of the found mean-level differences within the context of the pandemic. Although, such underlying processes must be further explored in future research. It is a strength of the study to have been able to measure students' protective factors and emotional problems prior to the pandemic and make comparisons during the pandemic.5Children are in many ways a vulnerable group. Our study demonstrates general positive adaptation during the COVID-19 pandemic among Swedish middle school students. Students' emotional problems showed no differences, whilst small differences in student's relationships to significant others and factors of psychological adjustment may be partially due to typical mean-level changes during development . MeaningThe PRISE study was approved by the Swedish Ethical Review Authority (reference number 2019–02755). The students’ legal guardians gave written informed consent. Students were orally informed about the research project, in a way as to facilitate understanding, and were informed of their right of withdrawal. The students gave oral informed consent on participating days.Emily G. Vira: Conceptualization, Formal analysis, Methodology, Writing – original draft, Visualization. Therése Skoog: Conceptualization, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – review & editing.None.